DNA binding to chromosomal DNA is essential for many fundamental biological processes including: transcriptional regulation, DNA replication and repair, recombination, and chromosome segregation. There is a fundamental gap in understanding how transcription factors (TF) bind regulatory regions located in compacted, high-order chromatin. Therefore, there is a fundamental need to determine the mechanistic rules defining TF binding to chromatin. The long-term goal for this project is to define the biological rules dictating TF binding to chromosomal DNA these rules include transcription factor binding site orientation within a nucleosome, DNA and histone modifications, cofactor and cooperative binding, and binding to subnucleosome particles. The overall objective of this application is to develop a high throughput approach to measure the principles of transcription factor binding to nucleosomal DNA. To accomplish this objective a new high-throughput next-generation sequencing assay will be developed to allow the simultaneous and quantitative examination of thousands of different nucleosomes in a single assay. The goal of this application will be accomplished by three specific aims: 1) Formation of a nucleosome library, 2) High-throughput protein-nucleosome binding assays with a library of nucleosomes, and 3) Define TF-nucleosome binding after histone modifications. Under the first aim, in vitro nucleosomes will be generated from thousands of in silica designed and naturally occurring DNA sequences in a single reaction, allowing a TF binding site to occur in all possible nucleosomal orientations with various neighboring sequence context. In the second aim, a new methodology, Pioneer-seq, will be developed where a transcription factor's binding affinity is determined to thousands of nucleosomes within a nucleosome library containing differing sequences, orientations, and variants. In the third aim, Pioneer-seq will be extended to examine how histone tail modifications amend TF-nucleosome binding. Overall, this project will develop a high- throughput quantitative method to determine binding principles for any protein to nucleosomal DNA in the presence or absence of histone modifications. This contribution will be significant because it can be applied to study many biological responses including: cell growth, regulation of cell-division, embryonic development, differentiation, response to environmental stresses, apoptosis and the development of a variety of disease states. In addition, biological principles can be addressed involving cooperative binding, binding to subnucleosomes, TF-histone interactions, and sequence content.